1. Field of the Invention
The present invention relates to novel cathepsin L-like enzyme extracted from northern shrimp (Pandalus eous), purification method thereof, polynucleotide encoding cathepsin L-like cysteine protease enzyme which is a novel protease newly identified from northern shrimp, polypeptide encoded by the same, and to the use of such polynucleotide and polypeptide.
2. Background Art
Protease is a generic term for enzymes that hydrolyze peptide bonds of proteins, and is widely spread among microorganisms, plants and animals. Numerous proteases having different catalyst groups and substrate specificity have been isolated. Their scope of application is also wide-ranging, and they are utilized, for example in modifiers of food products, detergents, cosmetic materials, clarifying agents for beer, tanning agents for leather, and medicaments.
Protease is one of the most important groups of enzymes which hydrolyze peptide bonds of protein, is widely spread among microorganisms, plants and animals, and is involved in various biological processes. In addition, proteases are categorized mainly into 4 families based on the catalyst groups: aspartic protease, cysteine protease, serine protease, and metalloprotease. The molecular action mechanism of these enzymes has been broadly investigated. SH protease (cysteine protease) having a SH group as active center includes enzymes such as bromelain. Cathepsins belonging to the papain superfamily of this cysteine protease are categorized into cathepsin L subfamily and cathepsin B subfamily. Cathepsin L subfamily includes cathepsins H, L, S, F, V, and W, and has 2 important motifs, i.e. ER(F/W)NIN motif and GNFD motifs which are dispersed motifs. The former ER(F/W)NIN motif does not exist in cathepsin B family and cathepsins C, O, and X.
In mammals, cathepsin L exists in lysosome and has potent endoprotease activity, although it has the characteristic that it does not show exo-type activity. Cathepsin L and cathepsin L-like cysteine proteases have so far been identified from several animals, and their sequences have been determined. These are listed below.
Bombyx mori (domestic silkworm)
    Yamamoto Y., Takimoto K., Izumi S., Toriyama-Sakurai M., Kageyama T., Takahashi S. Y. Molecular cloning and sequencing of cDNA that encodes cysteine proteinase in the eggs of the silkmoth, Bombyx mori. J. Biochem. 116(6):1330-1335(1994).Bos taurus (cow)    Unpublished.Drosophila melanogaster (fruit fly)    Tryselius Y., Hultmark D. Cysteine proteinase 1 (CP1), a cathepsin L-like enzyme expressed in the Drosophila melanogaster haemocyte cell line mbn-2. Insect Mol. Biol. 6(2): 173-181(1997).Homo sapiens (human)    Joseph L. J., Chang L. C., Stamenkovich D., Sukhatme V. P. Complete nucleotide and deduced amino acid sequences of human and murine preprocathepsin L. An abundant transcript induced by transformation of fibroblasts. J. Clin. Invest. 81(5):1621-1629(1988).Homarus americanus (American lobster)    Laycock M. V., MacKay R. M., Di Fruscio M., Gallant J. W. Molecular cloning of three cDNAs that encode cysteine proteinases in the digestive gland of the American lobster (Homarus americanus). FEBS Lett. 292:115-120(1991).Mus musculus (Mouse)    Portnoy D. A., Erickson A. H., Kochan J., Ravetch J. V., Unkeless J. C. Cloning and characterization of a mouse cysteine proteinase. J. Biol. Chem. 261:14697-14703(1986).Nephrops norvegicus (Norway lobster)    Le Boulay C., Van Wormhoudt A., Sellos D. Molecular cloning and sequencing of two cDNAs encoding cathepsin L-related cysteine proteinases in the nervous system and in the stomach of the Norway lobster (Nephrops norvegicus). Comp. Biochem. Physiol. 111:353-359(1995).Penaeus vannamei (Pacific white shrimp)    Le Boulay C., Van Wormhoudt A., Sellos D. Cloning and expression of cathepsin L-like proteinases in the hepatopancreas of the shrimp Penaeus vannamei during the intermoltcycle. J. Comp. Physiol. B 166:310-318(1996).Rattus norvegicus (Norway rat)    Ishidoh K., Towatari T., Imajoh S., Kawasaki H., Kominami E., Katunuma N. Suzuki K. Molecular clonig and sequencing of cDNA for rat cathepsin L. FEBS Lett. 223:69-73(1987).
With respect to collagenolytic activity of cathepsin L, it has been reported that cathepsin L of rat degrades collagen under acidic conditions at 37° C. Barrett, A. J. and Kirschke, H., Cathepsin B, Cathepsin H, and Cathepsin L., Methods Enzymol. 80, 535-561. (1981).